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PubMed1990

Structural requirements for C3d,g/Epstein-Barr virus receptor (CR2/CD21) ligand binding, internalization, and viral infection.

The structure of CR2, the human C3d,g/EBV receptor (CR2/CD21) consists of 15 or 16 60-70 amino acid repeats called short consensus repeats (SCRs) followed by a transmembrane and a 34-amino acid intracytoplasmic domain. Functions of CR2 include binding the human complement component C3d,g when it is covalently attached to targets or cross-linked in the fluid phase. In addition, CR2 binds the Epstein-Barr virus (EBV) and mediates internalization of EBV and subsequent infection of cells. In order to explore functional roles of the repetitive extracytoplasmic SCR structure and the intracytoplasmic domain of CR2, we have created truncated CR2 (rCR2) mutants bearing serial deletions of extracytoplasmic SCRs and also the intracytoplasmic tail. We then stably transfected these rCR2 mutants into two cell lines, murine fibroblast L cells and human erythroleukemic K562 cells. Phenotypic analysis of these expressed mutants revealed that 1) The C3d,g- and EBV-binding sites are found in the two amino-terminal SCRs of CR2, 2) expression of SCRs 3 and 4 is further required for high affinity binding to soluble cross-linked C3d,g, 3) the intracytoplasmic domain of CR2 is not required for binding C3d,g or EBV but is necessary for internalization of cross-linked C3d,g as well as for EBV infection of cells, 4) monoclonal anti-CR2 antibodies with similar activities react with single widely separated epitopes, and 5) no functional roles can yet be clearly assigned to SCRs 5-15, as rCR2 mutants not containing these SCRs show no major differences from wild-type rCR2 in binding or internalizing cross-linked C3d,g or mediating EBV binding and infection.

Animals↗
PubMed1990

Antibody-dependent enhancement of simian immunodeficiency virus (SIV) infection in vitro by plasma from SIV-infected rhesus macaques.

Plasma from two rhesus macaques (Macaca mulatta) experimentally infected with the simian immunodeficiency virus (SIV; isolate SIVmac251) enhanced SIVmac infection of a human CD4+ lymphoblastoid cell line, MT-2. Prechallenge plasma samples from these animals and serum from SIV-negative macaques did not enhance infection. Compared with controls, infection enhancement was characterized by the rapid appearance of syncytium formation (3 to 4 days sooner), reverse transcriptase release (10-fold increase), and cytopathic effect (60% cell killing). Enhancement of activity was dependent on the presence of diluted, fresh SIV-negative macaque serum as a source of complement. A requirement for complement was shown by the absence of enhancement in heat-inactivated serum and by dose-dependent inhibition of enhancement in the presence of polyclonal antibody to monkey complement component C3. Monoclonal antibody to CD4 (OKT4a) blocked enhancement completely, while monoclonal antibody to the human complement component C3d receptor CR2 (OKB7) reduced enhancement by greater than 50%, indicating a requirement for CD4 and CR2 in mediating this phenomenon. SIV infection-enhancing activity appeared in macaques soon after experimental inoculation (28 days). The titer increased over time and peaked just prior to the death of both macaques from opportunistic infections and lymphoma. In vitro SIV infection enhancement is nearly identical to the in vitro complement-mediated, antibody-dependent enhancing (C'-ADE) activity observed in human immunodeficiency virus-positive human sera (Robinson et al., Lancet i:790-794, 1988; Robinson et al., J. Acq. Immun. Def. Synd. 2:33-42, 1989). These observations validate the macaque-SIV model for studies of C'-ADE.

Animals↗
PubMed1987

Quantification of C3d in biological fluids by an enzyme-linked immunosorbent assay.

Using a commercial source of peroxidase-labelled anti-C3d antibody (Dakopatts), an enzyme-linked immunosorbent assay (ELISA) has been developed to quantify the complement fragment C3d. The technique enables the detection of C3d in plasma, urine and cerebrospinal fluid (CSF). The C3d-ELISA therefore provides a very sensitive technique for the evaluation of complement activation in biological fluids. In both plasma and urine the technique is able to discriminate between samples from normal controls and patients with rheumatoid arthritis in whom complement activation is known to occur. A good correlation was found between results obtained by ELISA and those by laser nephelometry (r = 0.91, P less than 0.0001). Microtitre plates pre-coated with anti-C3d antibody and subsequently stored at -70 degrees C retained the ability to perform in this assay. The sensitivity, short assay time and use of commercial reagents and pre-coated plates give this technique numerous potential applications in the evaluation of complement activation.

Arthritis, Rheumatoid↗
PubMed1989

Monoclonal anti-human C3d antibodies: stabilization of the alternative pathway C3 convertase.

IgG mouse monoclonal antibody (mAb) was prepared by fusion of spleen cells from mice immunized with human C3d (mAb:C3d) using syngeneic thymocytes as feeder cells. mAb:C3d was assessed for its effect on the stabilization of the cell-bound alternative pathway C3 convertase EAC3bBb. It bound to cell-bound C3b and stabilized C3bBb at 30 degrees in the presence of EDTA-GVB. The plasma protein H reduced the stabilization effect of the stabilized C3 convertase. These results suggest that binding of antibody to C3d may stabilize C3bBb. It seems likely that such antibody induces in C3b conformational change, which increases the C3bBb complex stability.

Antibodies, Monoclonal↗
PubMed1982

Influence of time, temperature and coagulation on the measurement of C3, C3 split products and C4.

Quantitative and qualitative immunoelectrophoretic analyses of circulating C3, C3 split products and C4 were performed in matched EDTA plasma and serum obtained from 5 normal subjects and stored for up to 48 h at room temperature (18 degrees C-22 degrees C) and 4 degrees C. Fluctuations in apparent levels of C3 were greater in serum than plasma stored at room temperature, a fall in levels seen by 24 h being followed by a significant increase. By contrast, levels of C3 did not alter if stored at 4 degrees C. C4 levels in both EDTA plasma and serum remained unchanged for 24 h, a slight decrease being seen at 48 h. Levels of C4 remained constant if samples were stored at 4 degrees C. Crossed immunoelectrophoresis revealed a significant progressive decrease in C3 levels and a simultaneous increase in C3c occurring after 4 h in serum and 8 h in EDTA plasma, stored at room temperature. In studies conducted at 4 degrees C, similar but delayed fluctuations were seen. A progressive and significant increase in C3d levels was seen in both plasma and serum samples stored at room temperature, levels rising to 276% (plasma) and 308% (serum) of levels seen at zero time. At 4 degrees C marginal increases in C3d levels only were observed. These results suggest that in vitro degradation of C3 and C4 are readily facilitated by temperature, time and coagulation, and that conditions of collection and storage of samples must be optimized for the accurate definition of activation of the complement cascade.

Adult↗
PubMed1981

Double-decker rocket immunoelectrophoresis for direct quantitation of complement C3 split products with C3d specificities in plasma.

A double-decker rocket immunoelectrophoresis (DD-RIE) method for direct quantitation of complement split products with C3d determinants in human plasma is described. The usefulness of the DD-RIE method for monitoring C3 activation has been assessed and compared with conventional crossed immunoelectrophoresis (CIE) for C3c determination in a patient with iatrogenic septic shock and patients with rheumatoid arthritis. In contrast with CIE the DD-RIE method is quantitative by reference to a standard curve based on an internal reference C3d preparation and its sensitivity and assay capacity are superior to CIE. All reagents and antibody preparation are commercially available and the production of standards is easy. No overlapping was observed between C3d values in plasma from healthy persons and patients with active classical rheumatoid arthritis. The DD-RIE is highly suitable for routine use in laboratories of clinical immunology.

Complement C3↗
PubMed1987

Heterogeneity of C4d and C3d and their complex formation with serum albumin.

High molecular weight (Mr around 175 KD) forms of C4d and C3d as well as free C4d and C3d (Mr about 40 KD) were demonstrable in normal human serum (NHS). Following in vitro C activation in NHS by delta IgG, the 40 KD C4d component increased markedly. By immunofixation it was shown that the high molecular forms of C4d and C3d reacted with biotinylated anti-human albumin IgG, whereas the 40 KD-free C4d and C3d fragments did not. Furthermore, the incorporation of anti-albumin IgG in the first dimensional gel in crossed immunoelectrophoresis caused retention of the 175 KD C4d component but not of free C4d. The 175 KD C4d had a distinctly higher electrophoretic migration velocity (post-albumin region) than the 40 KD C4d fragment. The C3d-, C4d-serum albumin complexes could not be dissociated by reducing agents (DTT, mercaptoethanol), a non-ionic detergent, or exposure to high and low ionic strength.

Complement Activation↗
PubMed1991

Tissue distribution of the C3d/EBV-receptor: CD21 monoclonal antibodies reactive with a variety of epithelial cells, medullary thymocytes, and peripheral T-cells.

The CD21 antigen has been described to represent CR2, the receptor for the complement fragment C3d and also the receptor for the Epstein-Barr virus (EBV). Monoclonal antibodies B2, HB5, and B-ly4 belong to the CD21 cluster, recognizing different epitopes of the CD21-molecule. Immunohistology of lymphoid tissues employing these antibodies showed the known staining of B cells and dendritic reticulum cells. Surprisingly, B2, but not HB5 or B-ly4, stained a distinct spot in the cytoplasm of a major proportion of medullary thymocytes, in almost all peripheral blood lymphocytes, and in a substantial amount of cells in T-cell areas of peripheral lymphoid tissues. This distinct cytoplasmic B2 staining was confirmed by immuno-electronmicroscopy. A similar B2+ cytoplasmic dot was observed in B-lymphoblastic lymphomas. Staining of non-lymphoid tissues showed reactivity with all three CD21 mAb with epithelial cells of skin, lung, esophagus, jejunum, colon, pancreas, tonsil, adrenal cortex, renal tubuli, and parotid glands, and with hepatocytes and tongue muscle. In addition, endothelial cells of small vessels showed B2 staining. One possible explanation for our results is, that apart from the presence of B cells and follicular dendritic cells, a CD21-molecule may be expressed by other cell types. However, a maybe more likely explanation may be that the recognized epitopes are not exclusively associated with the C3d/EBV-receptor, but also with other structures. In particular should the possibility be recognized of cross-reactivity with CR2-related proteins, encoded by the large gene family, to which CR2 belongs.

Antibodies, Monoclonal↗
PubMed1991

Persistent reduced solubilization of immune complexes in lepromatous leprosy patients with reactions.

It is held that immune complexes (IC) play a vital role in the pathogenesis of some of the reactions in leprosy. The complement system is known to solubilize and render IC innocuous. We have previously shown that patients undergoing lepra reactions had lowered complement-mediated IC solubilization (CMS). We, therefore, undertook a prospective study of untreated multibacillary leprosy patients and monitored their CMS levels sequentially while on therapy. In addition, the concentrations of the complement component C3d, immunoglobulins G, A and M, and circulating immune complexes (CIC) were also estimated. A total of 26 patients were included in the study and were investigated at 3-month intervals for 3 years. Thirteen of the 14 patients who did not develop reactions at all had normal CMS values, although all of them showed elevated CIC. From the inception of treatment, 10 of the 12 patients who developed lepra reactions had low CMS values which remained below normal levels even after evidence of complement activation disappeared and long after the subsidence of reaction. It is suggested that this defective CMS acts as a predisposing cause of lepra reactions.

Adult↗
PubMed1990

A novel strategy for targeting CD4+ PPD-reactive T cells against tumour cells using PPD monoclonal antibody heteroconjugates.

We have constructed PPD monoclonal antibody heteroconjugates specific for a tumour-associated antigen of C57BL/6 melanomas or for human complement component C3d fixed de novo to murine fibrosarcoma cells (MC6A). The ability of our heteroconjugates to target CD4+ PPD-reactive T cells against the appropriate tumour targets was then determined in vitro. Heteroconjugate-treated B16-F10 and MC6A tumour targets were both able to present PPD to the specific T cells, resulting in activation and concomitant lymphokine secretion. Secreted lymphokines were then demonstrated to cause significant tumour cytolysis and cytostasis in vitro. Preliminary experiments in vivo suggest that this targeting system may provide the basis for a future immunotherapeutic strategy.

Animals↗
PubMed1988

Analysis of C3 deposition and degradation on bacterial surfaces after opsonization.

C3b and iC3b, opsonic fragments of C3, interact with specific receptors on phagocytic cells. After bacterial opsonization, C3 fragments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blotting, and immunodetection. For bacteria opsonized in 50% pooled human serum (PHS), C3 deposition and cleavage to iC3b occurred rapidly. C3b, iC3b, and C3d made up 17%, 64%, and 19%, respectively, of the C3 on Staphylococcus aureus and 53%, 44%, and 2% respectively, on Escherichia coli. Residual C3b was refractory to factor I cleavage, an occurrence enabling alternative pathway activation to continue. C3 deposited was quantitated by enzyme-linked immunosorbent assay; with 50% PHS, greater than 50% and 90% of total C3 deposition occurred within 5 and 10 min, respectively. With a lower percentage of PHS, maximal deposition required up to 60 min and was not achieved in less than 10% PHS. Ester-bound fragments represented 34% and 82% of covalently bound C3 on S. aureus and E. coli, respectively.

Amides↗
PubMed1989

Alzheimer patients and Down patients: cerebral preamyloid deposits differ ultrastructurally and histochemically from the amyloid of senile plaques.

The preamyloid deposits found in the cerebral grey matter of Alzheimer patients and Down patients following immunostaining with anti-beta-protein antisera are neither birefringent following Congo red staining nor fluorescent after thioflavine S treatment. Further, they contain small amounts of alpha 1-antichymotrypsin, sulfated glycosaminoglycans and complement fraction C3d, but not the P component. As suggested previously, the material accumulated in these deposits may lack the molecular conformation responsible for the properties of amyloid fibrils. To test this hypothesis, we selected cortical samples from 6 Alzheimer and 4 Down patients for an electronmicroscopical study of senile plaques and of preamyloid deposits, both identified by indirect immunogold staining with anti-beta-protein antiserum. We observed the labelling of 4-8 nm wide amyloid fibrils in the plaque cores and of extracellular electrondense, flaky and irregularly distributed material in the preamyloid deposits. In the latter, amyloid fibrils were very rarely detected. These findings support the view that preamyloid deposits mostly contain amyloid precursors that are not yet organized in fibrils.

Alzheimer Disease↗
PubMed1987

Neutrophil lysosomal enzyme release and complement activation during cardiopulmonary bypass.

Complement activation and neutrophil degranulation were concomitantly studied during uncomplicated cardiopulmonary bypass (CPB). Plasma concentrations of complement factor C4, complement split product C3d, the neutrophil lysosomal enzyme elastase complexed with alpha 1-proteinase inhibitor (PI) and fibronectin were measured in 12 patients, C3d and elastase/PI increased significantly during CPB (volume-corrected results). The C3d rise was almost linear, whereas elastase/PI showed exponential increase. Mean elastase/PI and mean C3d concentrations at different times during CPB covaried closely. The study showed that during CPB neutrophil lysosomal enzyme release is intimately related to complement activation, although activation of the two systems may be caused by a common third activator within the extracorporeal circuit.

Adult↗
PubMed1987

Inhibition of interleukin 3 function by a fragment of the third component of complement.

A C3d-like (C3d-1) fragment of 33 kDa was isolated and its biological activity studied. The fragment was generated from guinea pig C3b by porcine pancreas kallikrein and purified by fast protein liquid chromatography. The C3d-like fragment inhibited interleukin (IL) 2-dependent T lymphocyte proliferation. The suppressive activity of the described C3d-1 fragment was not restricted to lymphocytes as targets but inhibited in addition the proliferation of a nonlymphocyte mast cell line which was strictly IL3-dependent in its proliferative capacity. Kinetic studies implied early stages of cellular proliferation to be influenced. Furthermore, the C3d-1 fragment was not only an inhibitor of cellular proliferation but was also a potent inducer of leukocytosis.

Animals↗
PubMed1992

Expression of the C3d-binding protein (CR2) from Candida albicans during experimental candidiasis as measured by lymphoblastogenesis.

The complement C3d-binding protein (CR2) of Candida albicans has been purified by immunoaffinity chromatography, and its specificity has been characterized by immunoblotting with monoclonal antibodies to the C. albicans CR2 and the mammalian CR2. Recent studies with immunoelectron microscopy indicated that the CR2 was expressed during a systemic infection in a murine model of candidiasis. As a continuation of these observations, the immunogenicity of the C. albicans CR2 was investigated in a lymphoblastogenesis assay. Lymph node cells as well as splenic lymphocytes from mice infected subcutaneously with viable blastoconidia of C. albicans reacted to the C. albicans CR2 to a significantly greater extent than did lymphocytes from uninfected mice (P less than 0.01). The maximum stimulation of splenic lymphocytes by the purified receptor occurred at a concentration of 0.54 micrograms of protein per ml after 72 h of incubation of lymphocytes and receptor. Also, splenocytes from infected or CR2-immunized mice exhibited significantly reduced responses to the T-cell-dependent mitogen phytohemagglutinin (P less than 0.01). These data indicate that lymphocytes from infected mice respond to the C. albicans CR2 in a lymphoproliferation assay to a greater extent than do lymphocytes from uninfected mice, indicating that the CR2 is expressed in vivo.

Animals↗
PubMed1984

A simple method for quantitative measurement of C3d in human plasma.

We describe a simple and reliable method for quantitating C3d in human plasma. The method rests on the finding that native C3 and its activation/inactivation product C3c bind to Concanavalin A, whereas C3d does not. Rocket affinoimmunoelectrophoresis with Con-A Sepharose incorporated into an intermediate gel permits quantitation of free C3d in 2-20 microliters aliquots of EDTA-plasma without any manipulation prior to sample application. Using this method, we found that the level of circulating C3d in plasma of 30 healthy donors was usually well below 3% of maximally convertable C3d.

Complement C3↗